High speed molecular sensing with nanopores

ABSTRACT

Described herein are methods and devices for capturing and determining the identity of molecules using nanopores. The molecules can be counted, sorted and/or binned rapidly in a parallel manner using a large number of nanopores (e.g., 132,000 nanopores reading 180 million molecules in 1 hour). This fast capture and reading of a molecule can be used to capture probe molecules or other molecules that have been generated to represent an original, hard to detect molecule or portions of an original molecule. Precise counting of sample molecules or surrogates for sample molecules can occur. The methods and devices described herein can, among other things, replace flow cytometers and other counting instruments (e.g., while providing increased precision and throughput relative to a flow cytometer). In some cases, the devices and methods capture and hold particular molecules or surrogates of molecules in the nanopores and then eject them into clean solution to perform a capture, sorting, and binning function similar to flow cytometers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/894,577, filed on Oct. 23, 2013, which is incorporated by referenceherein in its entirety.

BACKGROUND

Some applications in genomic analysis require the detection of copynumber variation. Pre-natal screening, for example, may determine ifcertain portions of Chromosome 13, 19, and 21 are duplicated or deletedin fetal free floating deoxyribonucleic acid (DNA). One way toaccomplish this is to enrich a whole genome sample for the specificregions on the select chromosomes (e.g., via PCR). PCR however canintroduce bias or errors in the product in several different ways,including disparities between the rates of enzymes or differencesbetween primer binding affinities to particular sites.

BRIEF SUMMARY OF THE INVENTION

Described herein are methods, devices and systems for capturing anddetermining the identity of molecules using nanopores. The molecules canbe counted, sorted and/or binned rapidly in a parallel manner using alarge number of nanopores (e.g., 132,000 nanopores reading 180 millionmolecules in 1 hour). This fast capture and reading of a molecule can beused to capture probe molecules or other molecules that have beengenerated to represent an original, hard to detect molecule or portionsof an original molecule. This can be used, for example, in the detectionof nucleic acid (e.g., DNA) polymorphisms, such as copy numbervariation. Precise counting of sample molecules or surrogates for samplemolecules can occur. The methods and devices described herein can, amongother things, replace flow cytometers and other counting instruments(e.g., while providing increased precision and throughput relative to aflow cytometer). In some cases, the devices and methods capture and holdparticular molecules or surrogates of molecules in the nanopores andthen eject them into clean solution to perform a capture, sorting, andbinning function similar to flow cytometers.

In an aspect, the disclosure provides a method for molecular countingand/or sorting, comprising: (a) providing an array of nanopores, whereinan individual nanopore of said array is individually addressable by anadjacent sensing electrode; (b) providing a plurality of markers thateach comprise nucleotides, wherein at least two of the nucleotideshybridize with a nucleic acid sample, and wherein the markers arecapable of being captured by the individual nanopore and identifiedusing the sensing electrode; and (c) capturing and identifying themarkers with the array of nanopores at a rate of at least about 1 markerper second per nanopore.

In some embodiments, the sensing electrode is operated in Faradaic mode.

In some embodiments, the sensing electrode is operated in non-Faradaicmode.

In some embodiments, the nucleic acid sample is derived from a patient.

In another aspect, the disclosure provides a method for molecularcounting and/or sorting, comprising: (a) providing an array ofnanopores, wherein an individual nanopore of said array is individuallyaddressable by an adjacent sensing electrode operated in non-faradaicmode; (b) providing a plurality of markers capable of being captured bythe individual nanopore and identified using the sensing electrode; and(c) capturing and identifying the markers with the array of nanopores ata rate of at least about 1 marker per second per nanopore.

In some embodiments, the markers are captured and identified at a rateof at least about four markers per second per nanopore.

In some embodiments, the plurality of markers comprise at least fourdifferent markers.

In some embodiments, the markers comprise tails having at least fourdifferent lengths.

In some embodiments, the markers are identified based on a voltage atwhich the markers leave the nanopore.

In some embodiments, the method further comprises releasing the capturedmarkers from the nanopore.

In some embodiments, the plurality of markers comprise markers to besorted, wherein the markers to be sorted are captured, identified andheld in the nanopores, and wherein markers other than the markers to besorted are captured, identified and released from the nanopores.

In some embodiments, the markers to be sorted are released as a groupand collected.

In some embodiments, the markers to be sorted are released as a groupwhen the ratio of the number of markers to be sorted divided by aremaining number of markers that are captured and identified by thenanopores increases above a threshold.

In some embodiments, the method further comprises quantifying markersthat comprise less than about 0.05% of the total number of markers.

In some embodiments, wherein said capturing and identifying comprisescapturing and identifying at least about 1 million markers per hour.

In some embodiments, the rate is at least about 100 million markers perhour.

In some embodiments, the rate is at least about 1 billion markers perhour.

In some embodiments, said capturing and identifying comprises countingand/or sorting at least about 8 different types of markers.

In some embodiments, said capturing and identifying comprises countingand/or sorting at least about 32 different types of markers.

In some embodiments, said capturing and identifying comprises countingand/or sorting at least about 100 different types of markers.

In some embodiments, said capturing and identifying comprises countingand/or sorting at least about 500 different types of markers.

In some embodiments, said capturing and identifying comprises countingand/or sorting at least about 500 different types of markers

In some embodiments, the array of nanopores is configured to have aplurality of regions capable of performing the method on differentsamples.

In some embodiments, the markers are identified based on a current thatflows through the individual nanopore and/or a voltage at which themarker leaves the nanopore.

In some embodiments, the markers each comprise a single stranded nucleicacid molecule attached to a bead.

In some embodiments, the markers are generated by: (a) hybridizing afirst probe to the nucleic acid sample; (b) hybridizing a second probeto the nucleic acid sample adjacent to the first probe; (c) ligating thefirst probe to the second probe to produce a combined probe; and (d)capturing the combined probe with a bead attached to an oligonucleotide,wherein the olignonucleotide hybridizes with the combined probe.

In some embodiments, the method further comprises determining copynumber variation of a nucleic acid sequence in the nucleic acid sample.

In some embodiments, the method further comprises detecting differencesin copy number that are less than or equal to about 0.05%.

In some embodiments, the method further comprises quantifying relativeRNA expression levels in the nucleic acid sample.

In some embodiments, the method further comprises performing an ELISAassay on the nucleic acid sample.

In some embodiments, the first probe comprises between about 20 andabout 50 nucleotides.

In some embodiments, the second probe comprises between about 20 andabout 50 nucleotides.

In some embodiments, the first probe comprises biotin.

In some embodiments, the bead is magnetic.

In some embodiments, the method further comprises concentrating themarkers adjacent or in proximity to the array of nanopores with amagnetic field.

In another aspect, the disclosure provides a method for sequencing,counting, and/or sorting molecules, comprising: (a) providing an arrayof nanopores, wherein an individual nanopore of said array isindividually addressable by an adjacent sensing electrode operated innon-faradic mode or faradaic mode; (b) providing a plurality ofmagnetically attractable beads each coupled to a molecule among aplurality of molecules to be sequenced, counted and/or sorted using thearray of nanopores;

concentrating the magnetically attractable beads in the vicinity of thearray of nanopores with a magnet; and (c) sequencing, counting and/orsorting the molecules with the array of nanopores.

In some embodiments, the magnetically attractable beads comprise metal.

In some embodiments, the magnetically attractable beads comprise apermanent magnetic material.

In some embodiments, the concentration of the magnetically attractablebeads prior to concentrating the magnetically attractable beads is atmost 100 femto-molar.

In some embodiments, the concentration of the magnetically attractablebeads prior to concentrating the magnetically attractable beads is atmost 10 femto-molar.

In some embodiments, the concentration of the magnetically attractablebeads near the array of nanopores is increased by at least 100-fold bysaid concentrating.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “FIG.” and “Figure” herein), of which:

FIG. 1 schematically shows the steps of the method;

FIGS. 2A, 2B and 2C show examples of nanopore detectors, where FIG. 2Ahas the nanopore disposed upon the electrode, FIG. 2B has the nanoporeinserted in a membrane over a well and FIG. 2C has the nanopore over aprotruding electrode;

FIG. 3 shows an array of nanopore detectors;

FIG. 4 shows the array of nanopores divided into two lanes;

FIG. 5 shows an example of a signal generated by marker entities (ormarkers) passing into a nanopore;

FIG. 6 shows an example of a compact sensor circuit;

FIG. 7 shows an example of using nanopores to count, sort or bin markerentities;

FIG. 8 shows an example of a marker entity and method for generation ofa marker entity;

FIG. 9 shows an example of a marker entity having a uni-directionalgate;

FIG. 10 shows an example of the identification of a marker entity basedon its fall-out voltage;

FIG. 11 shows an example of using the fall-out voltage to calibrate theapplied voltage;

FIG. 12 shows an example of a device and/or method for sorting andbinning molecular entities; and

FIG. 13 shows an example of a computer system that is programmed orotherwise configured to implement methods of the present disclosure.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “nanopore,” as used herein, generally refers to a pore, channelor passage formed or otherwise provided in a membrane. A membrane may bean organic membrane, such as a lipid bilayer, or a synthetic membrane,such as a membrane formed of a polymeric material. The membrane may be apolymeric material. The nanopore may be disposed adjacent or inproximity to a sensing circuit or an electrode coupled to a sensingcircuit, such as, for example, a complementary metal-oxide semiconductor(CMOS) or field effect transistor (FET) circuit. In some examples, ananopore has a characteristic width or diameter on the order of 0.1nanometers (nm) to about 1000 nm. Some nanopores are proteins. Alphahemolysin is an example of a protein nanopore.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. A nucleotide caninclude A, C, G, T or U, or variants thereof. A nucleotide can includeany subunit that can be incorporated into a growing nucleic acid strand.Such subunit can be an A, C, G, T, or U, or any other subunit that isspecific to one or more complementary A, C, G, T or U, or complementaryto a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C,T or U, or variant thereof). A subunit can enable individual nucleicacid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG,AC, CA, or uracil-counterparts thereof) to be resolved. In someexamples, a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), or derivatives thereof. A nucleic acid may besingle-stranded or double stranded.

The term “polymerase,” as used herein, generally refers to any enzymecapable of catalyzing a polymerization reaction. Examples of polymerasesinclude, without limitation, a nucleic acid polymerase or a ligase. Apolymerase can be a polymerization enzyme.

Methods and Devices

In an aspect, the disclosure provides methods and devices for molecularcounting and/or sorting comprises providing an array of nanopores, whereeach nanopore is individually addressable and disposed adjacent to asensing electrode. Individually addressable nanopores can each providetheir own electronic signal (e.g., using the sensing electrodes). Insome cases, the voltage applied to each individually addressablenanopore can be individually controlled. In some cases, the nanoporesare divided into groups, where various groups of nanopores areindividually addressable (e.g., provide a signal and/or can haveindividually applied voltages) with respect to each other.

The method can comprise providing a plurality of marker entities (also“markers” herein) capable of being captured and identified by thenanopores. The marker entities can be any molecule or molecular complexcapable of being captured and identified by the nanopores. Thedisclosure provides some examples of marker entities and molecularentities.

In some cases, the method comprises capturing and identifying the markerentities with the array of nanopores. The sensing electrodes can beoperated in a non-faradaic (or capacitive) sensing mode (e.g., where theelectrode and electrolyte do not perform a redox reaction). In someembodiments, the marker entities are captured and identified quickly(e.g., at a rate of at least about 1 marker entity per second pernanopore).

FIG. 1 shows an example of the steps of the method. In some cases, themarkers are generated from a nucleic acid sample. The nucleic acidsample can be extracted from an organism, tissue or cell. The markerentities can be prepared according to the methods described herein (see,e.g., FIG. 8 and the corresponding text). The marker entities can bedetected with the aid of a nanopore array.

The devices and methods of the present disclosure can be capable ofdetecting and/or counting several different marker entities (e.g., onthe same nanopore and/or in parallel on different portions of thenanopore array). The method can be capable of counting and/or sortingany suitable number of marker entities. In some cases, the method iscapable of counting and/or sorting about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 10, about 12, about 15, about 20, about25, about 30, or about 50 different types of marker entities. In somecases, the method is capable of counting and/or sorting at least about2, at least about 3, at least about 4, at least about 5, at least about6, at least about 7, at least about 8, at least about 10, at least about12, at least about 15, at least about 20, at least about 25, at leastabout 30, or at least about 50 different types of marker entities.

Nanopore Arrays

Provided herein are systems and methods for counting, binning andsorting with the aid of a nanopore. The nanopore may be formed orotherwise embedded in a membrane disposed adjacent to a sensingelectrode of a sensing circuit, such as an integrated circuit. Theintegrated circuit may be an application specific integrated circuit(ASIC). In some examples, the integrated circuit is a field effecttransistor or a complementary metal-oxide semiconductor (CMOS). Thesensing circuit may be situated in a chip or other device having thenanopore, or off of the chip or device, such as in an off-chipconfiguration. The semiconductor can be any semiconductor, including,without limitation, Group IV (e.g., silicon) and Group III-Vsemiconductors (e.g., gallium arsenide).

In some cases, as a marker entity flows through or adjacent to thenanopore, the sensing circuit detects an electrical signal associatedwith the marker entity. The marker entity may be a subunit of a largermolecule. The marker entity may be a byproduct of a nucleotideincorporation event or other interaction between a tagged nucleic acidand the nanopore or a species adjacent to the nanopore, such as anenzyme that cleaves a marker entity from a nucleic acid. The markerentity may remain attached to a nucleic acid. A detected signal may becollected and stored in a memory location, and later used to count themarker entities. The collected signal may be processed to account forany abnormalities in the detected signal, such as errors.

FIG. 2 shows an examples of a nanopore detector (or sensor) havingtemperature control, as may be prepared according to methods describedin U.S. Patent Application Publication No. 2011/0193570, which isentirely incorporated herein by reference. With reference to FIG. 2A,the nanopore detector comprises a top electrode 201 in contact with aconductive solution (e.g., salt solution) 207. A bottom conductiveelectrode 202 is near, adjacent, or in proximity to a nanopore 206,which is inserted in a membrane 205. In some instances, the bottomconductive electrode 202 is embedded in a semiconductor 203 in which isembedded electrical circuitry in a semiconductor substrate 204. Asurface of the semiconductor 203 may be treated to be hydrophobic. Asample having marker entities being detected goes through the pore inthe nanopore 206. The semiconductor chip sensor is placed in package 208and this, in turn, is in the vicinity of a temperature control element209. The temperature control element 209 may be a thermoelectric heatingand/or cooling device (e.g., Peltier device). Multiple nanoporedetectors may form a nanopore array.

With reference to FIG. 2B, where like numerals represent like elements,the membrane 205 can be disposed over a well 210, where the sensor 202forms part of the surface of the well. FIG. 2C shows an example in whichthe electrode 202 protrudes from the treated semiconductor surface 203.

In some examples, the membrane 205 forms on the bottom conductiveelectrode 202 and not on the semiconductor 203. The membrane 205 in sucha case may form coupling interactions with the bottom conductiveelectrode 202. In some cases, however, the membrane 205 forms on thebottom conductive electrode 202 and the semiconductor 203. As analternative, the membrane 205 can form on the semiconductor 203 and noton the bottom conductive electrode 202, but may extend over the bottomconductive electrode 202.

Nanopores may be used to count, sort or bin marker entities indirectly,in some cases with electrical detection. Indirect detection may be anymethod where a marker entity does not pass through the nanopore. Themarker entity may pass within any suitable distance from and/orproximity to the nanopore, in some cases within a distance such thatmarker entities are detected in the nanopore.

Byproducts of nucleotide incorporation events may be detected by thenanopore. “Nucleotide incorporation events” are the incorporation of anucleotide into a growing polynucleotide chain. A byproduct may becorrelated with the incorporation of a given type nucleotide. Thenucleotide incorporation events are generally catalyzed by an enzyme,such as DNA polymerase, and use base pair interactions with a templatemolecule to choose amongst the available nucleotides for incorporationat each location. In some cases, the marker entities are used tosequence a nucleic acid molecule.

The nanopores can form an array. The disclosure provides an array ofnanopore detectors (or sensors) for detecting marker entities. Withreference to FIG. 3, a plurality of marker entities may be detected onan array of nanopore detectors. Here, each nanopore location (e.g., 301)comprises a nanopore, in some cases attached to a polymerase enzymeand/or phosphatase enzymes. There is also generally a sensor at eacharray location as described elsewhere herein. Each of the nanopores canbe individually addressable.

The array of nanopores may have any suitable number of nanopores. Insome instances, the array comprises about 200, about 400, about 600,about 800, about 1000, about 1500, about 2000, about 3000, about 4000,about 5000, about 10000, about 15000, about 20000, about 40000, about60000, about 80000, about 100000, about 200000, about 400000, about600000, about 800000, about 1000000, and the like nanopores. The arraycan comprise at least 200, at least 400, at least 600, at least 800, atleast 1000, at least 1500, at least 2000, at least 3000, at least 4000,at least 5000, at least 10000, at least 15000, at least 20000, at least40000, at least 60000, at least 80000, at least 100000, at least 200000,at least 400000, at least 600000, at least 800000, or at least 1000000nanopores.

In some cases, a marker entity is presented or concentrated near ananopore (e.g., magnetically). A nanopore sensor adjacent to a nanoporemay detect an individual marker entity, or a plurality of markerentities. One or more signals associated with marker entities may bedetected and processed to yield an averaged signal.

Marker entities may be detected by the sensor as a function of time.Marker entities detected with time may be used to determine the identityof the marker entity, such as with the aid of a computer system (see,e.g., FIG. 13) that is programmed to record sensor data and generate thecount, sorting or binning functions from the data.

The array of nanopore detectors may have a high density of discretesites. For example, a relatively large number of sites per unit area(i.e., density) allows for the construction of smaller devices, whichare portable, low-cost, or have other advantageous features. Anindividual site in the array can be an individually addressable site. Alarge number of sites comprising a nanopore and a sensing circuit mayallow for a relatively large number of marker entities to be detected atonce. Such a system may increase the through-put and/or decrease thecost of counting, sorting or binning

A marker entity may be detected using a sensor (or detector) having asubstrate with a surface comprising discrete sites, each individual sitehaving a nanopore, and in some cases a polymerase attached to thenanopore and a sensing circuit adjacent to the nanopore. The system mayfurther comprise a flow cell in fluid communication with the substrate,the flow cell adapted to deliver one or more reagents to the substrate.

The surface comprises any suitable density of discrete sites (e.g., adensity suitable for determining marker entities in a given amount oftime or for a given cost). Each discrete site can include a sensor. Thesurface may have a density of discrete sites greater than or equal toabout 500 sites per 1 mm². In some embodiments, the surface has adensity of discrete sites of about 200, about 300, about 400, about 500,about 600, about 700, about 800, about 900, about 1000, about 2000,about 3000, about 4000, about 5000, about 6000, about 7000, about 8000,about 9000, about 10000, about 20000, about 40000, about 60000, about80000, about 100000, or about 500000 sites per 1 mm². In some cases, thesurface has a density of discrete sites of at least 200, at least 300,at least 400, at least 500, at least 600, at least 700, at least 800, atleast 900, at least 1000, at least 2000, at least 3000, at least 4000,at least 5000, at least 6000, at least 7000, at least 8000, at least9000, at least 10000, at least 20000, at least 40000, at least 60000, atleast 80000, at least 100000, or at least 500000 sites per 1 mm².

In some cases, the array of nanopores is configured to have a pluralityof regions (e.g., lanes) capable of performing the method on differentsamples. The samples can be different, or the sample can be divided intoseparate volumes with different assays performed on each volume.

In some cases, a plurality of wells (including any subset of the totalnumber of wells) comprises a common electrolyte pool. Each well can havea membrane with a nanopore disposed over it and a sensing electrodebelow or in the well. As shown in FIG. 4, the wells 401 may be separatedinto rows by walls 402 such that the row of wells shares a commonelectrolyte pool above the wells. Separating the biochip into sectionsas described here can allow multiple samples to be analyzed on a singlebiochip (e.g., by putting different samples in different sections of thechip).

Sensing Electrodes and Operation Thereof

The marker entities can be identified based on a current that flowsthrough the nanopore and/or a voltage at which the marker entity leaves(or is removed from) of the nanopore (e.g., fall out voltage). FIG. 5shows a prophetic plot where marker entities block the current flowingthrough the nanopore over time. The current is at a baseline level 505in the absence of a marker entity in the nanopore. The current can bereduced to different extents when different tags are located in thenanopore (e.g., 501, 502, 503, 504). Detection of marker entities basedon the fall out voltage is described below.

The current can be detected with a sensing electrode (e.g., which caninclude or be in electrical communication with a sensing circuit). Thesensing electrodes can be capable of either Faradaic or non-Faradaicsensing modes.

In Fardaic conduction mode, metal electrodes and a conductive salt canreact (e.g., perform an oxidation/reduction (redox) reaction) to form anew metal species and an electron that is later sensed by the chip'ssensor circuit. In Faradaic mode, a flow of ions can be generated by anapplied electrical potential between the electrodes, which can cause theelectrodes to react with ions in solution. In an example of silverchloride (AgCl) electrodes, an excess electron at one electrode under anapplied potential can cause chloride anion (Cl) to be expelled while alack of electrons at the other electrode can cause the silver (Ag)present to react with Cl⁻ and form AgCl. This system (e.g., reduction:electron+AgCl àAg(s)+Cl; oxidation: Cl⁻+Ag(s) à AgCl+electron) isdescribed as Faradaic and can be representative of any model using theoxidation and reduction of any metal to produce a flow of ions. Tomaintain a balance of Ag and AgCl at the electrodes and to help balanceions present on either side of a bilayer or membrane and nanopore as thesystem is operated, it may be necessary to occasionally (or frequently)reverse the potential on the electrodes to reverse the the reaction.

The flow of ions can also be generated by non-Faradaic means. Innon-Faradaic mode (also “capacitive mode” and “fast mode” herein), themetal electrode and the salt do not generally react (e.g., and do notperform a redox reaction). The result can be that the metal electrodedoes not generally form a new species. In non-Faradaic mode, a flow ofsalt ions can be established by applying a voltage (or electricalpotential) drop across a capacitive double layer existing between ametal and a salt or liquid. Under a potential, the capacitance of thedouble layer can be substantial enough such that the double layer canconduct and hold charge until the double layer (capacitor) reaches itsmaximum ability to store charge. Removing the potential and letting thecapacitor discharge through the nanopore can produce a flow of salt ionsthat can be detected by the sensor circuit. By switching the voltagefast enough (e.g., switching the polarity or magnitude of the voltage),a series of discharge cycles can be strung together that are closeenough in time to detect and represent the effects of moleculesinteracting with the nanopore. This technique has the benefit ofallowing a very small metal or non-metal conducting electrode to produceion and current flow without the electrode being degraded or changingover the course of the experiment.

Capacitive methods can be used to attract and repel ions to and from theelectrodes, but the ions may not cause a chemical reaction at thesurface of the electrodes. Electron flow can still be induced at theelectrodes; however it can be the result of charge influence, notphysical chemical reactions and electron ejection or capture. Themanipulation of charge and ionic flow in the non-Faradaic method alsocan benefit from occasionally (or frequently) reversing the potentialapplied to the electrodes; for example to reset a capacitance value tozero or substantially zero, or an undetectable limit.

Methods of the present disclosure can be implemented with Faradic metalelectrode nanopore arrays, however the non-Faradaic operation can resultin significantly faster counting and run for significantly longerperiods of time. In addition, the non-Faradaic approach can be the basisfor very fast attraction and capture of a marker entity. It can alsothen be used to repulse and or expulse a marker entity near or in thevicinity of a nanopore barrel.

In both Faradaic and non-Faradaic modes, the act of reversing thepotential can cause the marker entity in the nanopore to reversedirection. In nanopore systems, it can be difficult to take readings ofboth positive and negative currents. In the case of reading onlypositive currents, all negative applied potential readings can be readas zero. As a result, the position of the marker entity can be lost. Insome instances, the marker entity may even be ejected from the nanoporeand it can be necessary to re-capture the marker entity during the nextpositive applied potential.

Use of Faradaic or non-Faradaic electrical detection of marker entitiesusing electrodes of small or microscopic size (e.g., as is the case in amassively parallel nanopore array) can cause the marker entities toreverse direction. Molecules can be detected and polymers sequenced(marker entities can comprise polymers) in such a system by measuringthe flow of ions past a molecule being held or passing through ananopore. To create many pores and make parallel readings of manysimilar or different molecules, many electrodes and their associatednanopores can be used. To create many electrodes/nanopores in a smallarea, the electrodes may be small. Small electrodes and/or the smallamount of reagents in the side of electrodes sealed with a membrane orbilayer material can cause electrodes to lose their effectiveness intranslating ionic flow to electrical current.

FIG. 6 shows an example of a compact sensing circuit. An applied voltageVa can be applied to an opamp 600 ahead of a MOSFET current conveyorgate 601. Also shown here are an electrode 602 and the resistance of themarker entity detected by the device 603.

An applied voltage Va can drive the current conveyor gate 601. Theresulting voltage on the electrode is then Va−Vt where Vt is thethreshold voltage of the MOSFET. In some instances, this results inlimited control of the actual voltage applied to the electrode as aMOSFET threshold voltage can vary considerably over process, voltage,temperature, and even between devices within a chip. This Vt variationcan be greater at low current levels where sub-threshold leakage effectscan come into play. Therefore, in order to provide better control of theapplied voltage, an opamp can be used in a follower feedbackconfiguration with the current conveyor device. This can ensure that thevoltage applied to the electrode is Va, independent of variation of theMOSFET threshold voltage. In some cases, the voltage applied to theelectrode is calibrated using the fall-out voltage as described below.

The sensors and/or methods described herein can be operated innon-faradaic mode or in faradic mode. In some cases, including trehalosein the solution(s) allows faradic mode electrodes to operate for aboutan hour, which can be long enough to perform molecular counting and/orsorting as described herein. In some cases, faradic readings may givebetter resolution than non-faradaic operation.

Marker Entities and Detection Thereof

Methods of the present disclosure can include capturing and identifyingthe marker entities with the array of nanopores. The marker entities canbe any molecule or molecular complex, but in some cases they arepolymers (e.g., nucleic acids or peptides) attached to beads. FIG. 7shows an example of marker entities having different polymers (in thiscase two different markers 705 and 710) attached to beads 715. Thepolymer portion of the marker entities can be drawn into the nanoporewhere they block the current flowing through the nanopore. Eachdifferent type of marker entity can provide a unique electronicsignature, where a nanopore having no marker entity 720 is distinguishedfrom a nanopore having a first marker entity 725, which is distinguishedfrom a nanopore having a second marker entity 730.

The plurality of marker entities can comprise any number of differentmarker entities and/or the methods and devices described herein can becapable of distinguishing between any number of different markerentities. In some cases, the marker entities have different polymersattached to a bead (e.g., 705 vs. 710). Examples of different polymersinclude poly-ethylene glycol (PEG), nucleic acids having differentsequences, or peptides having different sequences. In some cases, themarker entities have the same polymers (e.g., both PEG) attached to abead, but the length of the polymer is varied (e.g., 705 vs. 735). Insome cases, the type of polymer can be identified based on a level ofcurrent (e.g., FIG. 5) and the length of the polymer can be identifiedbased on its fall-out voltage (e.g., FIG. 10).

The sample can have any number of different marker entities. In somecases, the sample has about 2, about 3, about 4, about 5, about 6, about7, about 8, about 10, about 12, about 15, about 20, about 25, about 30,or about 50 different types of marker entities. In some cases, thesample has at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 10, at least about 12, at least about 15, at least about 20,at least about 25, at least about 30, or at least about 50 differenttypes of marker entities.

The plurality of marker entities can comprise (polymer) tails having anynumber of different lengths. In some cases, the molecular entitiescomprise tails having about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, or about 10 different lengths. In some cases, themolecular entities comprise tails having at least about 2, at leastabout 3, at least about 4, at least about 5, at least about 6, at leastabout 7, at least about 8, at least about 9, or at least about 10different lengths.

In some cases, the marker entities comprise a single stranded nucleicacid molecule attached to a bead. The marker entities can be generatedin any suitable way.

With reference to FIG. 8, in some cases, the marker entities aregenerated by hybridizing a first probe 805 to a genomic DNA sample 810,hybridizing a second probe 815 to the genomic DNA sample adjacent to thefirst probe, and ligating 820 the first probe to the second probe toproduce a combined probe. In some cases, the first probe has a biotinmolecule attached 825. The second probe can have a sequence of bases 830(e.g., two bases) that provide a unique current level in the nanoporefor a given marker. In some cases, the second probe can determine thecurrent level, and by varying the length of the first probe, the lengthof the marker entity's tail can be varied to provide a second means ofdetermining the identity of the marker entity (e.g., current level andtail length via fall-out voltage). The second probe can hybridize to anoligonucleotide 835 that is attached to a bead 840 (e.g., for captureand isolation of the combined probe). The method can comprise capturingthe combined probe with a bead attached to an oligonucleotide, whereinthe olignonucleotide hybridizes with the combined probe.

The marker entities, first probes, second probes and/or combined probescan have any suitable length. In some cases, the marker entities, firstprobes, second probes and/or combined probes comprise nucleotides. Insome instances, the first probe comprises about 10, about 15, about 20,about 25, about 30, about 35, about 40, about 45, about 50, about 60,about 80, or about 100 nucleotides. In some instances, the first probecomprises at least about 10, at least about 15, at least about 20, atleast about 25, at least about 30, at least about 35, at least about 40,at least about 45, at least about 50, at least about 60, at least about80, or at least about 100 nucleotides. In some embodiments, the firstprobe comprises at most about 10, at most about 15, at most about 20, atmost about 25, at most about 30, at most about 35, at most about 40, atmost about 45, at most about 50, at most about 60, at most about 80, orat most about 100 nucleotides. In some cases, the first probe comprisesbetween about 20 and about 50 nucleotides.

In some instances, the second probe comprises about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about60, about 80, or about 100 nucleotides.

In some instances, the second probe comprises at least about 10, atleast about 15, at least about 20, at least about 25, at least about 30,at least about 35, at least about 40, at least about 45, at least about50, at least about 60, at least about 80, or at least about 100nucleotides. In some embodiments, the second probe comprises at mostabout 10, at most about 15, at most about 20, at most about 25, at mostabout 30, at most about 35, at most about 40, at most about 45, at mostabout 50, at most about 60, at most about 80, or at most about 100nucleotides. In some cases, the second probe comprises between about 20and about 50 nucleotides.

Since the first and second probes hybridize next to each other at aparticular site on sample DNA, the marker entities carry informationderived from the sample DNA. A slight temperature increase candissociate the combined probe from the sample. In some cases, this cycleis repeated until a region of interest of the DNA sample has multiplecombined probes (e.g., the sample can generate multiple marker entitiesand/or a marker entity can be formed from more than two probes).

For linear sequencing or for detection in a nanopore these individualcombined probes can have incorporated select molecules that allow theisolation of and subsequent linking of each of the combined probes intolong read strands (e.g., containing from 2 up to 10,000 or more combinedprobes). These combined probes in these read strands can be from onespecific sequence region from one sample, resulting a read strand ofrepeated identical combined probes. In some cases, these read strandscan be from multiple specific sequence regions from one sample resultingin a read strand containing a mixture of different (from 2 to 1,000 ormore) combined probes. One way to link these combined probes is to labelthe 5′ end of one and the 3′ end of the other. The labels can be anycombination of F4B and HiNyc (i.e., Solulink), streptavidin and biotin,or an alkyne and azide (i.e., click chemistry).

In some cases, strand mediated ligation can be used to ligate theindividual hybridized and ligated probes together into a single longstrand for single-loading and sequencing in a nanopore based system.Such a system may include ligation and separation of un-ligated probesand sample DNA from the desired ligated probes.

In addition, these read strands can be from multiple specific sequenceregions from multiple different samples resulting in read strandscontaining a mix of different (from 2 to 1,000s or more) combined probeswith each probe having a sample identifier or sample bar-codeincorporated.

Each combined probe can have any combination or number of features suchas; unique hybridization sections of probe molecules that bind adjacentto each other at a specific site selected for enrichment; non-bindingsection that identifies what sample number the full probe is from; andbiotin or other attachment molecule(s) that allows separation of desiredligated probes from un-ligated probes.

In some embodiments of the method, a modification of the polymer can bemade to allow the molecule to thread through the nanopore and yet beunable to reverse direction through the nanopore. This probe method canuse the incorporation of uni-directional gate sections (e.g., to allownon-enzymatic, strand sequencing of the product of this reaction onmassively parallel electrical detection, nanopore based systems).

In some cases, the marker entity is only capable of passing through thenanopore in one direction (e.g., without reversing direction). Themarker entity can have a hinged gate attached to the marker entity thatis thin enough to pass through the nanopore when the gate is alignedwith the marker entity tail in one direction, but not in anotherdirection. With reference to FIG. 9, the disclosure provides a markerentity molecule, comprising a first polymer chain 905 comprising a firstsegment 910 and a second segment 915, where the second segment isnarrower than the first segment. The second segment can have a widththat is smaller than the narrowest opening of the nanopore. The markerentity molecule can include a second polymer chain 920 comprising twoends, where a first end is affixed to the first polymer chain adjacentto the second segment and a second end is not affixed to the firstpolymer chain. The marker entity molecule is capable of being threadedthrough a nanopore in a first direction where the second polymer chainaligns adjacent to the second segment 925. In some cases, the markerentity molecule is not capable of being threaded through the nanopore ina second direction where the second polymer chain does not alignadjacent to the second segment 930. The second direction can be oppositethe first direction.

The first and/or second polymer chains can comprise nucleotides. In somecases, the second polymer chain base pairs with the first polymer chainwhen the second polymer chain does not align adjacent to the secondsegment. In some instances, the first polymer chain is affixed to a bead935.

The second segment can comprise any polymer or other molecule that isthin enough to pass through a nanopore when aligned with the gate(second polymer). For instance, the second segment can comprise a-basicnucleotides (i.e., a nucleic acid chain not having any nucleic acidbases) or a carbon chain.

The creation of the gate can be done in many ways. The molecule may besynthesized directly or the molecule can be appended or ligatedtogether. For example, a DNA strand can be created with and alkynelabeled nucleotide incorporated wherever a gate is to be attached. Asecond azide end-labeled nucleotide (e.g., that may be antisense to thenucleotide latch area) can be attached using click chemistry. Otherattachment chemistries and techniques maybe utilized includingcommercial methods (e.g., Solulink) or Amine-COOH combination.

Fall Out Voltage

The marker entities can be identified based on a voltage at which themarker entities are dislodged from or leave (or removed from) thenanopore (fall-out voltage). In non-Faradaic mode, marker entitieshaving tails (e.g., polymers) of various lengths can fall out of thenanopore at different voltages as the voltage decreases. FIG. 10 shows aplot of current through the nanopore (solid lines) and applied voltage(dashed lines) versus time. The current can decrease when a molecule iscaptured in the nanopore 1005. As the applied voltage is decreased overtime 1010, the current decreases until the molecule falls out of thenanopore, at which time the current increases to the expected level atthe applied voltage. The applied voltage at which the molecule falls outcan depend on the length of the molecule. For example, a marker entityhaving a 30 base tail can fall out around 40 milli-volts (mV) 1015,while a marker entity having a 50 base tail can fall out around 10 mV1020. As shown in this example, marker entities having tails shorterthan 30 bases can fall out of the nanopore at applied voltages higherthan 40 mV 1025.

Various current levels and fall-out voltages can be used to identifymarker entities. For example, the ability to detect 4 different currentlevels and 2 different fall-out voltages can allow the use of 8different marker entities.

In some cases, the applied voltage can be calibrated or re-calibratedusing the fall-out voltage. The calibration can permit theidentification of the marker entities. In some examples, calibrationincludes programming a computer processor of or associated with abiochip (or nanopore sensor apparatus) with known marker entities havingidentifiable signals, and storing the signals in a memory location of orassociated with the biochip for subsequent measurements.

Referring to FIG. 11, for a given marker entity having an averagefall-out voltage 1105, there can be variation 1110 in the fall-outvoltage for different nanopores or for different measurements on thesame nanopore over time. Adjusting this fall-out voltage to an expectedvalue can make the data easier to interpret and/or more accurate.

Molecule-specific output signals from single-molecule nanopore sensordevices can originate from the presence of an electrochemical potentialdifference across an ionically impermeable membrane surrounded by anelectrolyte solution. This trans-membrane potential difference candetermine the strength of the nanopore-specific electrochemical currentthat can be detected by electronics within the device via eithersacrificial (i.e., Faradaic) or nonsacrificial (i.e., non-Faradaic)reactions occurring at the electrode surfaces.

For any given state of the nanopore (i.e., open channel, captured state,etc.), the time-dependent trans-membrane potential can act as an inputsignal that can determine the resulting current flowing through thenanopore complex as a function of time. This nanopore current canprovide the specific molecular signal output by the nanopore sensordevice. The open-channel nanopore current can be modulated to varyingdegrees by the interactions between the nanopore and the capturedmolecules which partially block the flow of ions through the channel.

These modulations can exhibit specificity for the type of molecule thathas been captured, allowing some molecules to be identified directlyfrom their nanopore current modulations. For a given molecule type and afixed set of device conditions, the degree of modulation of theopen-channel nanopore current by a captured molecule of this type canvary depending on the trans-membrane potential applied, mapping eachtype of molecule to a particular current-vs.-voltage (I-V) curve.

Systematically variable offsets between the applied voltage settings andthe trans-membrane potential can introduce horizontal shifts of this I-Vcurve along the horizontal voltage axis, potentially reducing theaccuracy of molecular identification based on the measured currentsignals reported by the nanopore sensor device as an output signal.Therefore, uncontrolled offset between the applied and trans-membranepotentials can be problematic for accurately comparing measurements ofthe same molecule under the same conditions.

This so-called “potential offset” between the externally-appliedpotential and the actual trans-membrane potential can vary both withinand between experiments. Variations in potential offset can be caused byboth variations in initial conditions and by time-dependent variations(drift) in the electrochemical conditions within the nanopore sensordevice.

Removing these measurement errors can be done as described here bycalibrating the time-dependent offset between the applied voltage andthe trans-membrane potential for each experiment. Physically, theprobability of observing escape events of nanopore-captured moleculescan depend on the trans-membrane potential applied and this probabilitydistribution can be the same for identical samples of molecules underthe same conditions (e.g., the sample may be a mixture of differenttypes of molecules provided that their proportions do not vary betweensamples). In some cases, the distribution of voltages where escapeevents occur for a fixed sample type provides a measure of the offsetbetween the applied and trans-membrane potentials. This information canbe used in order to calibrate the applied voltage across the nanopore,eliminating systematic sources of error caused by potential offsetswithin and between experiments and improving the accuracy of molecularidentification and other measurements.

For a given nanopore sensor apparatus operated with the same molecularsample and reagents, the expected value of the distribution of escapevoltages can be estimated from a statistical sample of the singlemolecular escape events (although each individual event can be astochastic process subject to random fluctuations). This estimate can betime-dependent to account for temporal drift of the potential offsetwithin the experiment. This can correct for the variable differencebetween applied voltage settings and actual voltage felt at the pore,effectively “lining up” all the measurements horizontally when plottedin I-V space.

In some cases, potential (i.e. voltage) offset calibration does notaccount for current gain and current offset variations, which can alsobe calibrated for improved accuracy and reproducibility of nanoporecurrent measurements. However, potential offset calibration is generallydone prior to gain and offset correction to prevent errors in estimatingthe current gain and current offset variations, since these in turn caninvolve fitting current vs. voltage (I-V) curves, and the results ofthese fits are affected by variations in voltage offset (i.e., shiftingthe data left-to-right (horizontally) in I-V space can introduce errorsin current gain and current offset calibration).

In some cases, the applied voltage is calibrated. The calibrating caninclude estimating an expected escape voltage distribution versus timefor the sensing electrode. The calibration can then compute a differencebetween the expected escape voltage distribution and a reference point(e.g., an arbitrary reference point, such as zero). The calibration canthen shift the applied voltage by the computed difference. In somecases, the applied voltage decreases over time.

In some cases, a distribution of expected escape voltages versus time isestimated. In some instances, the reference point is zero volts. Themethod can removes detected variations in expected escape voltagedistribution. In some cases, the method is performed on a plurality ofindependently addressable nanopores each adjacent to a sensingelectrode.

In some embodiments, the presence of the marker entity in the nanoporereduces the current measured with the sensing electrode at the appliedvoltage.

In some instances, the calibration increases the accuracy of the methodwhen compared to performing the method without calibration. In somecases, the calibration compensates for changes in electrochemicalconditions over time. In some instances, the calibration compensates fordifferent nanopores having different electrochemical conditions in adevice having a plurality of nanopores. In some embodiments, thecalibration compensates for different electrochemical conditions foreach performance of the method. In some cases, the method furthercomprises calibrating variations in a current gain and/or variations ina current offset.

Fast, Precise and Accurate Counting

Provided herein are methods and devices for identifying and/or countingmarker entities using nanopores. In some cases, the identifying and/orcounting is fast, precise and/or accurate.

The marker entities can be identified and/or counted at any suitablerate. In some cases, the marker entities are identified and/or countedat a rate of about 0.2, about 0.5, about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 15, about20, or about 30 marker entities per second per nanopore. In some cases,the marker entities are identified and/or counted at a rate of at leastabout 0.2, at least about 0.5, at least about 1, at least about 2, atleast about 3, at least about 4, at least about 5, at least about 6, atleast about 7, at least about 8, at least about 9, at least about 10, atleast about 15, at least about 20, or at least about 30 marker entitiesper second per nanopore.

In some instances, the method and/or nanopore array is capable ofidentifying about 500,000, about 1 million, about 5 million, about 10million, about 50 million, about 100 million, about 500 million, orabout 1 billion marker entities per hour. In some cases, the methodand/or nanopore array is capable of identifying at least about 500,000,at least about 1 million, at least about 5 million, at least about 10million, at least about 50 million, at least about 100 million, at leastabout 500 million, or at least about 1 billion marker entities per hour.

The methods and devices described herein can be used to determine copynumber variation or relative RNA expression levels. In some cases, themethods are very precise (e.g., can detect very small differences incopy number variation or relative RNA expression levels). In someinstances, the method is capable of detecting differences in copy numberof about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%,about 3%, or about 5%. In some instances, the method is capable ofdetecting differences in copy number of less than about 0.01%, less thanabout 0.05%, less than about 0.1%, less than about 0.5%, less than about1%, less than about 2%, less than about 3%, or less than about 5%.

The methods and devices described herein can be used to perform analternative to an enzyme-linked immunosorbent assay (ELISA) (e.g.,quantify dilute or rare entities). In some cases, the device or methodis capable of quantifying marker entities that comprise about 0.01%,about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, orabout 5% of the total number of marker entities. In some instances, thedevice or method is capable of quantifying marker entities that compriseless than about 0.01%, less than about 0.05%, less than about 0.1%, lessthan about 0.5%, less than about 1%, less than about 2%, less than about3%, or less than about 5% of the total number of marker entities.

Sorting and Binning

The present disclosure provides devices and methods that can be used tosort the marker entities. In some cases, the sorted marker entities arecollected. The marker entities can be collected in separate reservoirsaccording to their identity (e.g., binned).

FIG. 12 shows an example of a device and/or method for sorting andbinning molecular entities. Nanopores not having a molecular entity aredepicted as open circles. Nanopores having a molecular entity to besorted and binned are depicted as circles filled with black. Nanoporeshaving a molecular entity other than the one to be sorted and binned aredepicted as circles filled with gray. The nanopores of the nanoporearray can capture and identify marker entities including ones to besorted 1205 and ones other than to be sorted 1210. In some cases, themolecular entities to be sorted are retained in the nanopore (e.g., bymaintaining a suitably high applied voltage). The molecular entitiesthat are other than the ones to be sorted can be expelled from thenanopore (e.g., by switching off or reversing the polarity of theapplied voltage), in some cases while still retaining the molecularentities to be sorted (e.g., because the nanopores are individuallyaddressable). The nanopores that do not have a captured marker entity tobe sorted can continue to capture, identify, and either retain or expelmarker entities based on their identity. During this continued process,additional marker entities to be sorted can be captured 1220. After anysuitable time and/or number of marker entities to be sorted have beencaptured and identified, most or all of the marker entities other thanthose to be sorted can be expelled 1225 to result in a nanopore arrayhaving all (or mostly all) marker entities to be sorted. At this point,most or all of the marker entities to be sorted can be expelled (e.g.,as a group) from the nanopore array. In some cases, the expelled markerentities to be sorted can be binned (e.g., as a group). In some cases,the method can be repeated to specifically capture a second group ofmarker entities to be sorted (e.g., other than the first group of markerentities to be sorted). In some cases, a first group of nanopores of thenanopore array capture and retain a first group of marker entities to besorted and a second group of nanopores of the nanopore array capture andretain a second group of marker entities to be sorted.

In some cases, the marker entities to be sorted are released as a groupwhen the percentage of marker entities to be sorted that are captured issuitably high. In some instances, the marker entities to be sorted arereleased when about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 99%, about 99.5%, or about 99.9% of the marker entitiesto be sorted are captured. In some cases, the marker entities to besorted are released when at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 99%, at least about 99.5%, or at least about 99.9%of the marker entities to be sorted are captured.

In some cases, the marker entities to be sorted are released as a groupwhen the ratio of marker entities to be sorted divided by markerentities other than the marker entities to be sorted that are capturedand identified by the nanopores decreases below a threshold. In somecases, the threshold is about 10%, about 5%, about 3%, about 1%, about0.5%, about 0.1%, about 0.05%, or about 0.01%. In some instances, thethreshold is less than about 10%, less than about 5%, less than about3%, less than about 1%, less than about 0.5%, less than about 0.1%, lessthan about 0.05%, or less than about 0.01%.

Magnetic Concentration

In some cases, the marker entities are at a low concentration in a bulksolution in contact with the nanopore array (e.g., below a concentrationat which the rate of capture by the nanopores is suitably high), but areconcentrated near the nanopores using magnetism (e.g., such that therate of capture and identification of the marker entities is suitablyhigh). In some cases, the bead portion of the marker entities (e.g., 935of FIG. 9) is magnetic or paramagnetic. In some cases, the methodcomprises concentrating the marker entities near the array of nanoporeswith a magnetic field, which can be provided by a magnet (e.g., apermanent magnet or an electromagnet).

In an aspect, a method for sequencing, counting, and/or sortingmolecules comprises providing an array of nanopores, where each nanoporeis individually addressable and disposed adjacent to a sensingelectrode. The method can also comprise providing a plurality ofmagnetically attractable (or active) beads coupled to a molecule to besequenced, counted and/or sorted using the array of nanopores andconcentrating the magnetically attractable beads in the vicinity of thearray of nanopores with a magnet. The method can further comprisesequencing, counting and/or sorting the molecules with the array ofnanopores.

In some cases, the magnetically attractable beads comprise metal. Insome instances, the magnetically attractable beads comprise a permanentmagnetic material.

The marker entities and/or magnetically attractable beads can be at anysuitably low initial concentration (e.g., in a bulk solution in contactwith the nanopore array) prior to concentrating the marker entitiesand/or magnetically attractable beads. In some cases, the initialconcentration is about 1 femto-molar (fM), about 5 fM, about 10 fM,about 50 fM, about 100 fM, about 500 fM, or about 1 micro-molar (μM). Insome cases, the initial concentration is at most about 1 femto-molar(fM), at most about 5 fM, at most about 10 fM, at most about 50 fM, atmost about 100 fM, at most about 500 fM, or at most about 1 micro-molar(μM).

The marker entities and/or magnetically attractable beads can beconcentrated near the nanopores to any suitable extent (e.g., a suitablyhigh ratio of the concentration near the nanopores after concentrationto the initial concentration in the bulk solution). In some cases, theconcentration of the magnetically attractable beads near the array ofnanopores is increased by about 5-fold, about 10-fold, about 50-fold,about 100-fold, about 500-fold, about 1000-fold, about 5000-fold, orabout 10000-fold. In some embodiments, the concentration of themagnetically attractable beads near the array of nanopores is increasedby at least about 5-fold, at least about 10-fold, at least about50-fold, at least about 100-fold, at least about 500-fold, at leastabout 1000-fold, at least about 5000-fold, or at least about 10000-fold.

Computer Systems

The devices described herein can be coupled to a computer system (e.g.,that collects data from and/or controls each of the individuallyaddressable nanopores). In some cases, the methods described herein areperformed with the aid of a computer system. The computer system caninclude one or more computer processors and a memory location coupled tothe computer processor. The memory location comprises machine-executablecode that, upon execution by the computer processor, implements any ofthe methods described herein.

FIG. 13 shows a system 1300 programmed or otherwise configured tocontrol or regulate one or more process parameters of a system of thepresent disclosure. The system 1300 includes a computer server(“server”) 1301 that is programmed to implement methods disclosedherein. The server 1301 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1305, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The server 1301 also includes memory 1310 (e.g.,random-access memory, read-only memory, flash memory), electronicstorage unit 1315 (e.g., hard disk), communication interface 1320 (e.g.,network adapter) for communicating with one or more other systems, andperipheral devices 1325, such as cache, other memory, data storageand/or electronic display adapters. The memory 1310, storage unit 1315,interface 1320 and peripheral devices 1325 are in communication with theCPU 1305 through a communication bus (solid lines), such as amotherboard. The storage unit 1315 can be a data storage unit (or datarepository) for storing data. The server 1301 can be operatively coupledto a computer network (“network”) 1330 with the aid of the communicationinterface 1320. The network 1330 can be the Internet, an internet and/orextranet, or an intranet and/or extranet that is in communication withthe Internet. The network 1330 in some cases is a telecommunicationand/or data network. The network 1330 can include one or more computerservers, which can enable distributed computing, such as cloudcomputing. The network 1330, in some cases with the aid of the server1301, can implement a peer-to-peer network, which may enable devicescoupled to the server 1301 to behave as a client or a server. The server1301 can be coupled to a system 1335 either directly or through thenetwork 1330. The system 1335 can be configured to perform nucleic acid(e.g., DNA, RNA) or polymeric (e.g., protein) sequencing or molecularcounting.

The storage unit 1315 can store process parameters (e.g., calibrationparameters) of the system 1335. The process parameters can includecharging and discharging parameters. The server 1301 in some cases caninclude one or more additional data storage units that are external tothe server 1301, such as located on a remote server that is incommunication with the server 1301 through an intranet or the Internet.

The server 1301 can communicate with one or more remote computer systemsthrough the network 1330. In the illustrated example, the server 1301 isin communication with a remote computer system 1340. The remote computersystem 1340 can be, for example, a personal computers (e.g., portablePC), slate or tablet PC (e.g., Apple® iPad, Samsung® Galaxy Tab),telephone, Smart phone (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistant.

In some situations, the system 1300 includes a single server 1301. Inother situations, the system 1300 includes multiple servers incommunication with one another through an intranet and/or the Internet.

Methods as described herein can be implemented by way of machine (orcomputer processor) executable code (or software) stored on anelectronic storage location of the server 1301, such as, for example, onthe memory 1310 or electronic storage unit 1315. During use, the codecan be executed by the processor 1305. In some cases, the code can beretrieved from the storage unit 1315 and stored on the memory 1310 forready access by the processor 1305. In some situations, the electronicstorage unit 1315 can be precluded, and machine-executable instructionsare stored on memory 1310. Alternatively, the code can be executed onthe second computer system 1340.

The code can be pre-compiled and configured for use with a machine havea processer adapted to execute the code, or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Aspects of the systems and methods provided herein, such as the server1301, can be embodied in programming. Various aspects of the technologymay be thought of as “products” or “articles of manufacture” typicallyin the form of machine (or processor) executable code and/or associateddata that is carried on or embodied in a type of machine readablemedium. Machine-executable code can be stored on an electronic storageunit, such memory (e.g., read-only memory, random-access memory, flashmemory) or a hard disk. “Storage” type media can include any or all ofthe tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless links, optical links or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

Various parameters of the system described herein can be presented to auser on a user interface (UI) of an electronic device of the user.Examples of UI's include, without limitation, a graphical user interface(GUI) and web-based user interface. The UI (e.g., GUI) can be providedon a display of an electronic device of the user or server 1301. Thedisplay can be a capacitive or resistive touch display. Such displayscan be used with other systems and methods of the disclosure.

The following examples are intended to illustrate, but not limit, theinvention.

EXAMPLES Example 1 Creation of a Non-Faradaic Array and Demonstration ofMarker Detection

A nanopore sensor chip is created with electrode metal optimized fornon-Faradaic, fast mode (e.g., non-Faradaic) operation. The electrodeprocessing results in an individual electrode with a capacitance of 20femto-Farads (fF) to 60 fF. A non-Faradaic conducting salt solution istested and selected to provide a repeatable and appropriate open channelcurrent level. The salt solution enables voltages sufficient to readilycapture free floating marker entities. The hardware and software of thenanopore sensor chip is modified for fast mode operation. Continuousoperation of the nanopore sensor chip is demonstrated with a minimum of5 active pores for 30 minutes. The pores capture (e.g., DNA) markers ata rate of at least 2 per second. Markers are tested and 4 differentmarkers are selected that give 4 different current levels with 95% orgreater accuracy over 1,800 marker captures. The test is replicated 5times.

Example 2 Optimization and Characterization of the Chip

Operating nanopores are created that are capable of operating in fastmode. A minimum of forty pores are created in five out of tenconsecutive attempts. Pores are created that are capable of operating infast mode and have a minimum of twenty pores that last for a minimum ofthirty minutes. The chip is operated in fast mode and demonstrates thecounting of marker entities. A solution containing two different markersis read with at least twenty pores operating in fast mode. Markers areread at a rate of two markers per second across twenty pores for thirtyminutes for a total of 72,000 reads. The anticipated read ratios arechecked with the expected ratios. The fallout voltage for four differentmarkers is characterized to determine if marker length can be used toincrease the potential pool of markers.

Example 3 Fast Molecular Sensing

A nanopore array having 264 individually addressable nanopores isprovided. About 75 of the nanopores are operating for the purpose ofsequencing. A mixture of four different marker entities is provided. Anoperating nanopore captures and identifies the marker entities at a rateof about four marker entities per second. The nanopore array reads about300 marker entities per chip per second, about 18,000 marker entitiesper chip per minute, or about 1,080,000 marker entities per chip perhour. In two hours, the nanopore array reads about 2,160,000 markerentities per chip.

Example 4 Fast Molecular Sensing

A nanopore array having 264 individually addressable nanopores isprovided. About seventy five of the nanopores are operating. A mixtureof eight different marker entities is provided with some of the markershaving different tail lengths. The nanopore captures and identifies themarker entities at a rate of about one per second per operatingnanopore. The nanopore array reads about seventy five marker entitiesper chip per second. The nanopore array reads about 4,500 markerentities per chip per minute, about 270,000 marker entities per chip perhour, or about 540,000 marker entities per chip in two hours.

Example 5 Fast Molecular Sensing

A nanopore array having 132,000 individually addressable nanopores isprovided. About 50,000 of the nanopores are operating. A mixture of fourdifferent marker entities is provided. The nanopore captures andidentifies the marker entities at a rate of about four per second peroperating nanopore. The nanopore array reads about 200,000 markerentities per chip per second, about 12,000,000 marker entities per chipper minute, or about 720,000,000 marker entities per chip in one hour.

Example 6 Fast Molecular Sensing

A nanopore array having 132,000 individually addressable nanopores isprovided. About 50,000 of the nanopores are operating. A mixture ofeight different marker entities is provided with some of the markershaving different tail lengths. The nanopore captures and identifies themarker entities at a rate of about one per second per operatingnanopore. The nanopore array reads about 50,000 marker entities per chipper second, about 3,000,000 marker entities per chip per minute, orabout 180,000,000 marker entities per chip in one hour.

Example 7 Fast Molecular Sensing

A nanopore array having 132,000 individually addressable nanopores isprovided. The array is divided into four lanes each having about 20,000nanopores. Each lane has about 7,500 operating nanopores and is capableof performing a different assay. A mixture of thirty two differentmarker entities is provided. The mixture is divided amongst the fourlanes. The nanopore captures and identifies the marker entities at arate of about four per second per operating nanopore. The nanopore arrayreads about 30,000 marker entities per lane per second, about 18,000,000marker entities per lane per minute, or about 108,000,000 markerentities per lane in one hour.

Example 8 Fast Molecular Sensing

A 96 well plate is populated with beads such that each well has beadsthat capture one marker. The markers that are created from one samplehave unique binding sites that allow them to bind to a specific bead.The mix of markers can be configured so that for each bead, eightdifferent markers can bind. The remaining markers have binding sites forother beads. The entire marker mix is created so that only 8 markers areallowed for each different bead. In this example, there are 8 markersfor each bead and 96 beads for 768 unique markers separated into groupsof 8 per well.

The sample solution is sequentially exposed to each well, drawing themagnetic beads to the bottom of the well after each exposure and movingthe sample solution to the next well for exposure. A collection ofmarkers can be separated for detection using the nanopore detectiontechnique described here. Other methods of spatially separating thebeads to allow for the separate collection markers can be performed(e.g., beads serially exposed to one solution containing markers, orbeads spatially positioned at known locations on a nanopore array chip).

The collection of markers in each well of the 96 well plate can bemelted off the bead or left on the bead and flowed through a channel onthe nanopore detector chip. The marker can be flushed from the flow cellafter detection in the flow cell and the next different collection ofmarkers attached to a different bead can be loaded and detected orcounted. The complete flushing of beads and markers can be assisted bythe magnetic properties of the beads. Applying a magnetic attractionforce as well as liquid washing force can help insure the completerinsing of nearly all markers from a flow cell.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for molecular counting and/or sorting,comprising: a. providing an array of nanopores, wherein an individualnanopore of said array is individually addressable by an adjacentsensing electrode; b. providing a plurality of markers that eachcomprise nucleotides, wherein at least two of the nucleotides hybridizewith a nucleic acid sample, and wherein the markers are capable of beingcaptured by the individual nanopore and identified using the sensingelectrode; and c. capturing and identifying the markers with the arrayof nanopores at a rate of at least about 1 marker per second pernanopore.
 2. The method of claim 1, further comprising releasing thecaptured markers from the nanopore.
 3. The method of claim 1, whereinthe plurality of markers comprise markers to be sorted, wherein themarkers to be sorted are captured, identified and held in the nanopores,and wherein markers other than the markers to be sorted are captured,identified and released from the nanopores.
 4. The method of claim 3,wherein the markers to be sorted are released as a group and collected.5. The method of claim 3, wherein the markers to be sorted are releasedas a group when the ratio of the number of markers to be sorted dividedby a remaining number of markers that are captured and identified by thenanopores increases above a threshold.
 6. The method of claim 1, whereinthe array of nanopores is configured to have a plurality of regionscapable of performing the method on different samples.
 7. The method ofclaim 1, wherein the markers are identified based on a current thatflows through the individual nanopore and/or a voltage at which themarker leaves the nanopore.
 8. The method of claim 1, wherein themarkers each comprise a single stranded nucleic acid molecule attachedto a bead.
 9. The method of claim 1, wherein the markers are generatedby: a. hybridizing a first probe to a nucleic acid sample; b.hybridizing a second probe to the nucleic acid sample adjacent to thefirst probe; c. ligating the first probe to the second probe to producea combined probe; and d. capturing the combined probe with a beadattached to an oligonucleotide, wherein the olignonucleotide hybridizeswith the combined probe.
 10. The method of claim 9, further comprisingdetermining copy number variation of a nucleic acid sequence in thenucleic acid sample.
 11. The method of claim 9, further comprisingquantifying relative RNA expression levels in the nucleic acid sample.12. The method of claim 9, further comprising performing an ELISA assayon the nucleic acid sample.
 13. The method of claim 9, wherein the firstprobe comprises biotin.
 14. The method of claim 9, wherein the bead ismagnetic.
 15. The method of claim 14, further comprising concentratingthe markers adjacent or in proximity to the array of nanopores with amagnetic field.
 16. A method for molecular counting and/or sorting,comprising: a. providing an array of nanopores, wherein an individualnanopore of said array is individually addressable by an adjacentsensing electrode operated in non-faradaic mode; b. providing aplurality of markers capable of being captured by the individualnanopore and identified using the sensing electrode; and c. capturingand identifying the markers with the array of nanopores at a rate of atleast about 1 marker per second per nanopore.
 17. A method forsequencing, counting, and/or sorting molecules, comprising: a. providingan array of nanopores, wherein an individual nanopore of said array isindividually addressable by an adjacent sensing electrode operated innon-faradic mode or faradaic mode; b. providing a plurality ofmagnetically attractable beads each coupled to a molecule among aplurality of molecules to be sequenced, counted and/or sorted using thearray of nanopores; c. concentrating the magnetically attractable beadsin the vicinity of the array of nanopores with a magnet; and d.sequencing, counting and/or sorting the molecules with the array ofnanopores.
 18. The method of claim 17, wherein the magneticallyattractable beads comprise metal.
 19. The method of claim 17, whereinthe magnetically attractable beads comprise a permanent magneticmaterial.
 20. The method of claim 17, wherein the concentration of themagnetically attractable beads near the array of nanopores is increasedby at least 100-fold by said concentrating.